Download Promoter-trapping in Saccharomyces cerevisiae

ã 2002 Oxford University Press
Nucleic Acids Research, 2002, Vol. 30, No. 24 e136
Promoter-trapping in Saccharomyces cerevisiae by
radiation-assisted fragment insertion
Markus Kiechle1,2, Palaniyandi Manivasakam1, Friederike Eckardt-Schupp2,
Robert H. Schiestl1 and Anna A. Friedl2,3,*
1
Department of Pathology, School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095,
USA, 2Institute of Molecular Radiation Biology, GSF-Research Center, D-85764 Neuherberg, Germany and
3
Radiobiological Institute, University of Munich, D-80336 Munich, Germany
Received October 9, 2002; Revised and Accepted October 19, 2002
ABSTRACT
Non-homologous insertion (NHI) of DNA fragments
into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast
Saccharomyces cerevisiae, the ef®ciency of NHI is
very low. Here we report that its ef®ciency can be
increased by g-irradiation of recipient cells at the
time of transformation. Radiation-assisted NHI
depends on YKU70, but its ef®ciency is not
improved by inactivation of RAD5 or RAD52. In a
pilot study, we generated 102 transformant clones
expressing a lacZ reporter gene under standard
conditions (30°C, rich medium). The site of insertion
was determined in a subset of eight clones in which
lacZ expression was altered by UV-irradiation. A
comparison with published data revealed that three
of the eight genes identi®ed in our screen have not
been targeted by large-scale transposon-based
insertion screens. This suggests that radiationassisted NHI offers a more homogeneous coverage
of the genome than methods relying on transposons
or retroviral elements.
INTRODUCTION
Random insertional mutagenesis is a powerful tool in the
analysis of gene function that is amenable to genome-wide
analyses and that has been applied in a variety of organisms
(1± 4). In addition to studying the phenotype associated with
disruption of the target sequence, by insertion of cassettes
containing appropriate elements it is also possible to conduct
gene expression analyses and to insert tag sequences that can
be used for protein isolation or localisation studies. Insertion
cassettes are often derived from mobile DNA elements, such
as retroviruses or transposons, and ef®cient insertion into
genomic DNA is then mediated by the respective mobilising
apparatus (e.g. integrases or transposases). As most mobile
elements exhibit a certain degree of target site speci®city,
uniform coverage of all genes in the genome may be dif®cult
to achieve with this approach. For example, by insertional
mutagenesis using Tn3-derived mini-transposons a large
collection of Saccharomyces cerevisiae mutant clones has
been established (5,6). Although, until the end of the year
2001, the site of insertion has been characterised in more than
22 000 insertion clones, less than two-thirds of the about 6200
yeast genes are represented in this collection (7). In addition to
gene-size dependent biases in targeting ef®ciency, nonrandom insertion of Tn3-derived transposons (8) and unequal
representation of genes in the yeast library mutagenised may
account for this effect.
To achieve complete coverage of the genome, the introduction of complementary approaches is necessary. One such
approach is non-homologous insertion (NHI; 9) of DNA
cassettes not derived from mobile elements, a process which
appears to exploit the cellular machinery that normally repairs
DNA double-strand breaks (DSB) by non-homologous end
joining (NHEJ; 10). Successful application of NHI strategies
in large-scale insertional mutagenesis projects has, for
example, been described in mammalian cells, plants and
Schizosaccharomyces pombe (11±13). Similar approaches in
S.cerevisiae have been precluded by the low ef®ciency of NHI
in this organism (14).
We sought to establish an ef®cient NHI-based method for
random insertion of a promoter-trap cassette containing a lacZ
reporter gene in S.cerevisiae. Previous work demonstrated that
NHI ef®ciency increases upon co-transformation of restriction
endonucleases (14±16). Restriction-enzyme-mediated integration (REMI) was subsequently used for insertion mutagenesis
in a variety of organisms (17). To avoid a sequence bias
towards genomic recognition sites of the co-transformed
endonuclease, we here investigate whether g-irradiation is
suitable for facilitating NHI. Within the yeast genome, DSB
induced by sparsely ionising radiation appear to be distributed
randomly (18), although small-scale variations due to radical
shielding by DNA-associated proteins cannot be excluded.
Irradiation should therefore result in a largely random
distribution of insertion sites. Here, we report that g-irradiation
shortly before or after the heat-shock step of transformation
enhances the rate of NHI suf®ciently to allow its application in
a promoter-trap scheme. To demonstrate proof-of-principle,
we analysed 2000 NHI transformants for lacZ expression.
Among the 102 lacZ expressing clones eight transformants
*To whom correspondence should be addressed at Radiobiological Institute, University of Munich, Schillerstrasse 42, 80336 Munich, Germany.
Tel: +49 89 5996807; Fax: +49 89 5996 840; Email: [email protected]
e136 Nucleic Acids Research, 2002, Vol. 30, No. 24
were isolated that exhibited altered lacZ expression after UVirradiation. Interestingly, three of the eight insertion sites were
located in genes that so far have not been found hit in the more
than 22 000 clones obtained in the large-scale transposonbased mutagenesis screen (5±7). Thus, radiation-assisted NHI
of linearised plasmids may be suitable as a complementary
approach in order to allow full coverage of the S.cerevisiae
genome.
MATERIALS AND METHODS
For optimising radiation-assisted NHI, plasmids pM150 and
pM151 (16) were used. These pUC18 derivatives contain the
URA3 gene and differ in the restriction sites present in the
MCS. For constructing plasmid pFA6-kanMX6-lacZ, a 3.1 kb
BamHI/SalI fragment containing the lacZ gene was isolated
from plasmid pCM159 (19) and inserted in BamHI/SalIdigested plasmid pFA6-kanMX6 (20). Plasmid MKM20 was
generated by PCR ampli®cation of the lacZ gene, using pFA6kanMX6-lacZ as a template, with primers lacZ-C-BamHI (5¢CGA ATT CGG ATC CAG CTG AAG CTT CGT ACG-3¢)
and lacZ-N-BglII (5¢-CGA ATT CAG ATC TAC TGG CCG
TCG TTT TAC-3¢), and insertion of the BamHI/BglII-digested
product into the BglII site of pM151. The lacZ allele thus
obtained lacks the ®rst 20 nucleotides, including the start
codon, and lacZ expression is obtained only upon in-frame
insertion of the BglII-linearised plasmid MKM20 into
expressed genes.
NHI experiments were performed in haploid strain RSY12
(MATa leu2-3,112 his3-11,15 ura3D::HIS3), in which the
entire URA3 open reading frame was replaced by the HIS3
gene (14). Strain RSY12 rad5 was generated by transformation of a rad5D::kanMX-lacZ deletion cassette generated by
PCR, using primers rad5-kanMX-C (5¢-CTA TTC AAA CAG
CAT CTG GAT TTC TTC AAT TCT CCT TTT TCT GGG
TCA CCC GGC CAG CG-3¢) and rad5-lacZ-N (5¢-ATG AGT
CAT ATT GAA GAA AGG AAG TTT TTT AAC GAT CCC
GTC GTT TTA CAA CG-3¢) on template pAF6-kanMX6lacZ. Generation of strains RSY12 yku70 and RSY12 rad52
has been described earlier (15,21).
Yeast cells were transformed using a high ef®ciency
method (22). For transformation, plasmids pM151 or
MKM20 were digested to completion with BglII, extracted
with phenol, precipitated and dissolved in sterile water.
Control transformations, using the same batch of cells, were
performed with circular plasmid YEplac195 (23). For
radiation-assisted NHI, transformation reaction mixes were
g-irradiated at room temperature, using a 60Co-g-source
(Atomic Energy of Canada, Ltd) at a dose rate of 10 Gy
min±1. If not stated otherwise, irradiation took place immediately after the heat-shock step of the transformation procedure.
Relative transformation frequencies were determined by
normalising the rate of transformation of the linearised
plasmid (per microgram of DNA) relative to the rate of
transformation with the control plasmid.
Integrated plasmids and adjacent genomic sequences were
recovered by plasmid rescue as described (21), except that
several restriction enzymes that do not cut within the plasmid
sequence (BamHI, BspEI, NheI and XhoI) were used in
parallel digestions of each DNA sample, in order to enhance
the ef®ciency of plasmid rescue. Plasmids with ¯anking
PAGE 2 OF 6
chromosomal DNA, as identi®ed by migration behaviour in
agarose gels, were sequenced by Medigenomix GmbH
(Martinsried, Germany) using sequencing primers P1 (5¢AGC GGA TAA CAA TTT CAC ACA GGA-3¢) and P2 (5¢ACT CCA GCC AGC TTT CC-3¢). Sequencing data were
analysed by a BLASTN search on the Saccharomyces
Genome Database (SGD; http://genome-www.stanford.edu/
Saccharomyces/).
To identify lacZ-expressing transformants, patches of cells
on selection plates were covered with ~10 ml chloroform and
incubated for 5 min to permeabilize cells. After removing the
chloroform, plates were air-dried for 5 min, before they were
covered with X-Gal suspension (1 mg/ml X-Gal in 1% LMPagarose solution, dissolved at 60°C). After solidi®cation of the
agarose, plates were incubated at 30°C up to 24 h to allow for
colour development. To quantify UV-induced lacZ expression, cells were grown to logarithmic phase (5 3 106 cells/ml),
harvested by centrifugation and re-suspended at a titre of 1 3
108 cells/ml in liquid holding recovery (LHR) buffer (67 mM
K2HPO4/KH2PO4, 100 mM glucose, pH 5). Aliquots of 2 ml
were UV-irradiated (254 nm) in the dark in plastic Petri dishes
under constant stirring. Post-irradiation incubation of irradiated suspensions was performed in the dark at 30°C under
constant shaking. At given time points, 1 ml aliquots were
removed, and after centrifugation cells were re-suspended in
0.5 ml Z-buffer (75 mM Na2HPO4, 50 mM NaH2PO4, 10 mM
KCl, 1 mM MgSO4) and immediately frozen at ±80°C, where
they were kept until detection. For detection, all samples of an
experimental series were thawed simultaneously and each
50 ml of the suspensions were used to determine the OD600.
The remaining cells were incubated for 1 h at 37°C with 100 ml
zymolyase solution (zymolyase T100, Seikagaku Corp.,
Japan; 0.5 mg in 1 ml Z-buffer). After completion of lysis,
100 ml of CPRG solution (4 mg/ml Chloro-Phenol-RedGalactopyranosid in Z-buffer) were added and incubated at
room temperature. Incubation times (tinc) varied between 1 and
180 min, according to the different levels of lacZ expression.
Reactions were stopped by adding 200 ml 1 M Na2CO3. After
15 min centrifugation at 15 000 r.p.m., the optical density of
the supernatant was measured at 574 nm and 634 nm. The
b-galactosidase activity in Miller units was calculated as:
[MU] = [(OD574 ± OD634) 3 1000] / (OD600 3 tinc). Semiquantitative screening of lacZ expression was performed
similarly, except that cell titres at the time of irradiation were
not determined.
RESULTS
The frequency of NHI events increases upon irradiation
We investigated the in¯uence of irradiation on the frequency
of NHI of BglII-linearised plasmid pM151 (16), which
contains the yeast URA3 gene, into the genome of strain
RSY12, in which the URA3 locus had been deleted (14). The
frequency of transformation with plasmid pM151 was
normalised with respect to parallel control transformations
with a circular episomal plasmid. g-Irradiation of cells
immediately after the heat-shock step of the transformation
procedure caused an increase in NHI frequency of up to
~15-fold (Fig. 1A). Thus, about 150 transformants can be
obtained in one transformation experiment, using 7 mg of
PAGE 3 OF 6
Figure 1. Frequency of transformation with linearised plasmid pM151 when
cells were treated with g-irradiation immediately after the heat-shock step of
the transformation procedure. The number of transformants per microgram
plasmid DNA was normalised with respect to parallel control transformations with circular plasmid YEplac195. (A) Comparison of wild-type strain
RSY12 (white) and its rad5 (light grey) and yku70 (dark grey) mutant
derivatives. Indicated are means and standard deviations from three independent experiments (except RSY12 rad5, 200 Gy: one experiment).
(B) Comparison of RSY12 [white; same data as in (A)] and its rad52 (grey)
mutant derivative. Note that the scale of the y-axis has changed. Data for
RSY12 rad52 are from two experiments (0 and 50 Gy) or one experiment
(100 and 200 Gy).
linearised plasmid. We refer to this method as radiationassisted NHI. Comparable effects were seen when cells were
irradiated immediately before the heat shock, while the effect
declined with increasing duration of the period between
irradiation and transformation, until, with periods exceeding 4
h, no positive effect of irradiation could be seen anymore (data
not shown). Additional experiments using plasmids linearised
with other enzymes showed that radiation-assisted NHI is
more pronounced with fragments terminating in singlestranded overhangs than with plasmids terminating in blunt
ends (data not shown).
An increase of NHI frequency with irradiation is not seen in
Ku70-de®cient mutants [Fig. 1A; (21)], demonstrating that the
insertion of transformed fragments into broken chromosomal
DNA involves NHEJ. In general, NHEJ contributes little to the
repair of radiation-induced chromosomal breaks in yeast (10).
We hypothesised that the ef®ciency of radiation-induced NHI
may be increased in mutant strains that exhibit more potent
NHEJ. For example, the Rad5 protein, inactivation of which
confers moderate radiosensitivity, has been invoked in the
regulation of DSB repair pathways in S.cerevisiae. While
wild-type cells repair plasmids gapped within a sequence that
is also present on a chromosome almost exclusively by
homologous recombination with the chromosomal donor
sequence, rad5 mutants rely predominantly on NHEJ-type
repair of these plasmids (24). In contrast to its in¯uence in
plasmid repair, however, the rad5 mutation does not appear to
Nucleic Acids Research, 2002, Vol. 30, No. 24 e136
have a role in NHI, as, both with and without irradiation, the
relative transformation ef®ciency was not signi®cantly altered
in rad5 mutants as compared with the wild-type strain
(Fig.1A).
It has been proposed that NHEJ processes and homologous
recombination competes for repair of DNA DSB and that the
decision of which pathway is used is, at least in part,
determined by the proteins initially binding to the DNA ends
(25). Thus, by inactivation of Rad52, a DNA end-binding
protein essential for homologous recombination, more ends
may become accessible to Ku proteins and the relative
frequency of NHEJ events may increase. Indeed, as compared
with wild-type cells, we observe a strong increase in the
relative frequency of NHI events in rad52 mutants both with
and without irradiation (Fig. 1B). However, since the control
transformation with circular plasmids is about 50 times less
ef®cient in the rad52 mutant than in wild-type [(4.2 + ±3.6) 3
102 versus (1.9 + ±0.5) 3 104 transformants/mg plasmid DNA
in rad52 mutants and wild-type, respectively], the absolute
number of transformants is reduced in the mutant. Reduced
transformation with circular plasmids in rad52 mutant strains
has been observed previously (15). In addition, the mutant is
much more sensitive to the cell killing effects of radiation
(survival of 14 versus 45% at 100 Gy), further reducing the
number of transformants obtainable.
Application of radiation-assisted NHI in a promotertrapping scheme
The bacterial b-galactosidase gene lacZ is a widely used
reporter gene, the expression of which can easily be monitored
qualitatively and quantitatively (26). Insertion of a BglII/
BamHI-digested PCR fragment encompassing the lacZ gene
into the BglII site of pM151 created plasmid MKM20. The
lacZ allele used in our studies lacks the ®rst 25 nucleotides
including the start codon. By targeted in-frame insertion into
the chromosomal RAD54 gene, a gene well known for its UV
inducibility (27), we veri®ed the suitability of this allele to
serve as a reporter gene in analysis of UV-induced gene
induction (data not shown).
To use for promoter trapping, plasmid MKM20 was
digested with BglII and transformed into strain RSY12 using
a high ef®ciency transformation protocol. Immediately after
the heat-shock step, the cells were irradiated with 50 Gy. A
dose of 50 Gy was chosen to achieve both suf®cient cell
survival (~60%) and high ef®ciency of NHI. At this dose, on
average 2 DSB/cell are induced (18). Transformation ef®ciency with plasmid MKM20 was comparable with that of
pM151. A total of about 2000 Ura+ transformants were
generated and tested by X-Gal overlay for in-frame insertion
of MKM20 into yeast genes expressed under standard growth
conditions (YPD, 30°C). Detectable expression of the inserted
lacZ gene was found in 102 transformants (5%). To identify
those transformants in which the reporter gene expression was
affected by UV-irradiation, a semi-quantitative screen was
conducted. Cells grown to logarithmic growth phase were resuspended in non-growth buffer, half of the sample was
UV-irradiated (60 J/m2) and the other half served as mocktreated control. The non-growth condition was chosen so that
changes in gene expression are not due to any secondary effect
of radiation-induced cell cycle arrest. Cells were lysed 2 h
after treatment and b-galactosidase activity was determined
e136 Nucleic Acids Research, 2002, Vol. 30, No. 24
PAGE 4 OF 6
Table 1. Insertion of the lacZ reporter relative to the trapped gene in
clones displaying differential expression in a semi-quantitative screen after
UV-irradiation
Clone ID
Gene
Insertion of lacZ N-terminus
MK12
MK33
MK35
MK38
MK42
MK60
MK66
MK82
APG1
FIR1
MRP8
SDS24
UBC13
NVJ1
BUD4
YBR052c
In-frame within ORF
In-frame within ORF
In-frame within ORF
In-frame within ORF
In-frame in intron
In-frame within ORF
22 bp upstream
In-frame within ORF
using a colourimetric assay. Out of 102 lacZ-expressing
clones, 12 candidates differing at least 2-fold in expression in
treated versus untreated cells were identi®ed and chosen for
further analysis. In eight of these, the site of insertion of the
promoter-trapping construct was successfully determined by
plasmid rescue (see Table 1). In the remaining clones, plasmid
rescue and/or sequencing were not successful (three cases) or
the construct was found to be fused to a fragment from the
2 mm plasmid which obviously enabled autonomous replication (one case).
In six of the eight events indicated in Table 1, insertion of
the reporter construct occurred in-frame within a coding
region. In clone 42, insertion occurred in-frame within the
intron of the UBC13 gene, 46 bp upstream of the branch-point
(28). In clone 66, insertion occurred 22 bp upstream of the
BUD4 initiator codon. The next in-frame ATG codon is
located at position ±165 (as counted from the BUD4 initiator
codon); since translational frameshift events occur readily in
yeast (6), alternatively an out-of-frame ATG at position ±36
may have been used to allow expression of the reporter gene.
To investigate whether insertion events differ from events
observed spontaneously, we sequenced both insertion junctions (Fig. 2). Similar to events seen in spontaneous NHI (9),
in the majority (6/8) of the insertion events at least one
junction apparently was facilitated by microhomology between plasmid end and genomic sequence. In ®ve of the eight
integrations, no or only one nucleotide of the genomic
sequence was lost. Only one event (clone 60) was associated
with a large genomic rearrangement (inversion or duplication).
An analysis of the kinetics of expression of the lacZ-fusion
proteins in response to UV irradiation with 60 J/m2 veri®ed
strong induction (>2-fold) for fusions involving MRP8,
SDS24, NVJ1 and YBR052c, and 1.5-fold induction for
APG1 (Fig. 3). These genes have been found by microarray
analyses to be generally stress-inducible (29±31). For the
fusion involving UBC13, b-galactosidase activity in UVirradiated and mock-treated samples differed by >1.5-fold, but
instead of showing a clear induction, the treated samples rather
exhibited a slower decline of b-galactosidase activity during
the post-treatment incubation than the mock-treated samples
(Fig. 3). In microarray and northern analyses, however,
increased amount of UBC13 transcripts after DNA damage
have been described (29±33). We assume that the different
conditions during post-treatment incubation (non-growth
versus growth conditions) are responsible for the discrepancies. A slightly accelerated decline of b-galactosidase activity
Figure 2. Junctions between plasmid MKM20 and genomic DNA, as determined by plasmid rescue and sequencing with primers P1 and P2. Remnants
of the single-stranded overhangs present in the transformed plasmid after
restriction digest are indicated in bold. For clari®cation, the ®rst nucleotides
of genomic sequence at the junction sites are labelled by shading. Regions
of microhomology at the junctions are underlined; nucleotides that are
inserted after plasmid integration are framed. Numbers in the chromosomal
sequences refer to chromosomal positions as indicated in the SGD database.
Note that in clone MK66, 16 nt of plasmid sequence were absent at the P1
junction.
in treated versus untreated sample was observed during posttreatment incubation in a clone involving a fusion with BUD4,
a gene which by others was described as repressed in response
to DNA damage due to cell cycle arrest (30). In contrast to the
results of the semi-quantitative analysis, no signi®cant difference between treated and untreated samples was observed
concerning the b-galactosidase activity of FIR1-lacZ fusions
when assaying the kinetics of expression.
Of the genes affected by insertion, only UBC13 is known to
be involved in response to DNA damage. To test whether the
other genes have an as yet uncharacterised function in damage
resistance, UV and g-ray sensitivity was tested for all insertion
clones. None of the clones (except for the ubc13 mutant)
exhibited increased sensitivity (data not shown), con®rming a
recent report that only a very small proportion of the genes
whose expression is altered after DNA damaging treatments
actually is involved in conferring resistance to these treatments (34).
DISCUSSION
The method used to insert a mutagenising fragment into
the genome may be a critical factor for successful application of insertional mutagenesis screens. Use of mobile
PAGE 5 OF 6
Figure 3. LacZ expression in samples treated with 60 J/m2 UV radiation
(®lled squares) and in mock-treated samples (open squares) as a function of
post-irradiation incubation under non-growth conditions. LacZ activity is
indicated in Miller units.
element-derived mechanisms is highly ef®cient, but often
accompanied by a target bias that precludes uniform coverage
of all genomic loci. The alternative method, random insertion
of fragments without using mobilising machinery, is still
poorly characterised in terms of mechanistic details. Recent
evidence supports the notion that NHI is mediated by
components of the NHEJ pathway of DSB repair (15,21,35;
this work). Here we demonstrate that radiation-assisted NHI,
similar to REMI (9), results in an increased yield of
transformants. Optimal enhancement of NHI was seen when
irradiation took place around the time of the heat-shock step in
transformation, i.e. the time when the transformed DNA enters
the cell. Similar to spontaneous and restriction enzymemediated NHI, radiation-assisted NHI works better on fragments terminating in single-stranded overhangs than in bluntended fragments. Also, the junction sites observed in
radiation-assisted NHI were very similar to those observed
after spontaneous or enzyme-mediated NHI in their dependence on microhomologies and generation of small deletions or
insertions. Whether large genomic alterations (as seen in clone
60) occur more often in radiation-assisted NHI than in
spontaneous NHI (13) remains to be tested. Our attempts to
Nucleic Acids Research, 2002, Vol. 30, No. 24 e136
manipulate the regulatory system of DSB repair by inactivating RAD5 or RAD52 did not improve radiation-assisted NHI.
In line with our results, it has recently been found that
impairment of homologous recombination does not enhance
NHEJ ef®ciency in yeast (36). Whether, as suggested by the
data of these authors, the frequency of radiation-assisted NHI
is increased in G1/G0 cell populations as compared with
logarithmically growing populations, remains to be tested.
Expression of the lacZ reporter gene under standard growth
conditions (30°C, YPD) was observed in 5% of our
transformants. This is about the frequency expected, considering the proportion of coding DNA in S.cerevisiae [about
0.7; (37)], the probability of in-frame insertion in the correct
orientation (0.33 3 0.5), the frequency of essential genes
[0.19; (38)] and the fact that expression of some genes under
standard conditions may be too low to allow detection. Among
the clones expressing lacZ, ~8% were found to increase
expression upon UV-irradiation. Considering the experimental differences, this value compares well with results obtained
in expression studies using microarrays (29±31,34). Our
investigation differs from these studies in that we performed
post-irradiation incubation of treated samples and mocktreated controls under non-growth conditions. In addition,
depending on the site of insertion of the reporter construct, the
amount of lacZ-fusion proteins may not only re¯ect promoter
activity, but, at least in part, also post-transcriptional and posttranslational regulation of expression. It is known that in many
cases protein levels do not correlate with mRNA abundance
(39).
The most interesting aspect of this pilot study is that three of
the eight trapped genes identi®ed (FIR1, BUD4, MRP8) have
not yet been targeted in a large-scale transposon-based
insertional mutagenesis screen in spite of the impressive
number of more than 22 000 insertion events analysed so far
(5±7). We infer this from the fact that these genes are not listed
in the TRIPLES database [http://ygac.med.yale.edu/triples/
triples.htm; (7)], which describes all insertional mutagenesis
events generated by the transposon-based approach. Another
two of the genes trapped in our study (NVJ1, UBC13) have
been targeted only once by transposon-based trapping. In
contrast, many other genes, including APG1, SDS24 and
YBR052, have by now been hit multiple times. We conclude
that by radiation-assisted NHI a more complete coverage of
the genome can be obtained and that this method is suitable to
complement any existing large-scale mutagenesis screen.
A major concern may, however, arise from using a
clastogenic agent that has a potential of introducing additional
genetic and genomic alterations. At present, we cannot
estimate how often the analysis of insertion-associated
phenotypes will suffer from artefacts caused by radiationinduced additional alterations. It should be noted, however,
that good experimental practice requires that in any insertional
mutagenesis screen, including transposon-based screens,
phenotypes be carefully checked for segregation with given
insertion alleles.
We propose that radiation-assisted NHI is a suitable means
to complement existing large-scale studies in functional
genomics in S.cerevisiae and any other organisms that exhibits
a low-ef®ciency NHEJ system. It would, of course, be
desirable to use multi-purpose fragments in transformation
e136 Nucleic Acids Research, 2002, Vol. 30, No. 24
that allow, after insertion, to simultaneously investigate a
variety of endpoints.
ACKNOWLEDGEMENTS
We gratefully acknowledge expert technical assistance by
K. Winkler, ®nancial support by Deutsche Forschungsgemeinschaft (KI 796/1) and European Commission (FIS51999-00066), and travel grants by the Neuherberger
ForschungsfoÈrderung.
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